Total internal reflection illumination apparatus and microscope using this total internal reflection illumination apparatus

ABSTRACT

A total internal reflection illumination apparatus applied to a microscope which illuminates a sample through an objective having a numerical aperture enabling total internal reflection illumination, comprises a first total internal reflection mirror which is arranged in the vicinity of an outermost peripheral part of an observation optical path of the microscope to reflect an incident illumination light in a direction of the objective, a second total internal reflection mirror which is arranged at a symmetrical position with the first total internal reflection mirror to sandwich an observation optical axis and reflects return light reflected on a surface of the sample in a direction different from the illumination optical path, and a return light dimming part configured to dim the return light reflected by the second total internal reflection mirror.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based upon and claims the benefit of priorityfrom the prior Japanese Patent Applications No. 2001-341007, filed Nov.6, 2001; and No. 2001-374426, filed Dec. 7, 2001, the entire contents ofboth of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a total reflection illuminationapparatus applied to a microscope which illuminates a sample through anobjective having a numerical aperture enabling total internal reflectionillumination, and a microscope using this total internal reflectionillumination apparatus.

[0004] 2. Description of the Background Art

[0005] In recent years, functional analysis of a biological cell hasbeen vigorously carried out. In such function analysis of cells, inorder to observe a function of a cell membrane in particular, attentionis drawn to a total internal reflection fluorescence microscopy (TIRFM)which acquires total internal reflection fluorescence images from thecell membrane and a nearby part.

[0006] In such a total internal reflection fluorescence microscopy(which will be referred to as a “microscope” hereinafter), a totalinternal reflection illumination which locally illuminates only a sample(which may be also referred to as a “specimen” in some cases) in thevicinity of the glass surface is used. In this microscope, when theillumination light is totally reflected on the interface between thecover glass and the specimen, a fluorescent material is excited by usingthe light called evanescent light which permeates in a small range ofnot more than several 100 nm on the specimen side. Therefore, only thefluorescence in a small range in the vicinity of the cover glass isobserved. Accordingly, the background (scattered light or the like) isvery dark, and the weak fluorescence can be observed (for example,observation of the fluorescence of one molecule of the fluorescent dye).

[0007] Meanwhile, in the fluorescence observation by using such a totalinternal reflection illumination, a permeation depth of the evanescentlight which permeates from the glass surface to the sample side variesdepending on a refractive index of the glass and others. Further, thispermeation depth means a depth along which observation has been carriedout, and it also varies depending on a purpose of a speculum user.

[0008] Thus, there is also considered varying an incidence angle of theillumination light from the glass to the sample in accordance withconditions of the specimen or a depth along which observation should becarried out.

[0009] Meanwhile, in case of performing fluorescence observation usingthe total internal reflection illumination, there is known a microscopewhich conducts the total internal reflection illumination whichilluminates the sample through an objective.

[0010] For example, in such a microscope, a mirror which reflects thelight from a light source to an objective side is moved, and anincidence position of the illumination light to the objective iscontinuously moved in a direction away from the optical axis of theobjective. As a result, the incidence angle from the glass to the sampleis continuously changed and the incident-light fluorescence illuminationand the total internal reflection illumination are switched (see Jpn.Pat. Appln. KOKAI Publication No. 09-159922). It is to be noted that amicrometer or the like is generally used for movement of the mirrorwhich reflects the illumination light, namely, adjustment of theincidence angle from the glass to the sample because fine adjustment isrequired.

[0011] Furthermore, in another microscope, a frame of the objective hasa dual structure consisting of an inner frame and an outer frame, thelight from the light source is reflected by an annular mirror so thatthe illumination light can pass between the inner frame and the outerframe in the dual structure. A sample is illuminated with that light,and the return light from the sample is observed through the objective(see Jpn. Pat. Appln. KOKAI Publication No. 10-96861).

[0012] In the above-described structure, in the total internalreflection illumination which performs illumination through theobjective, the illumination light with which the specimen is irradiatedreturns to the objective in principle. Moreover, the mirror used to leadthe illumination light to the objective side is provided in anobservation optical path extending from the objective to observingmeans.

[0013] Therefore, the observation optical path of the fluorescenceemitted from the specimen crosses the illumination light or the totallyreflected return light. Therefore, the self-fluorescence generated onthe illumination light or the totally reflected return light beam entersthe observation light beam, and there is a possibility that afluorescence observation image may be deteriorated.

[0014] In addition, since the illumination light and the totallyreflected return light also cross each other, an interference fringe maybe generated due to crossing of the laser beams when the laser beam isused as the illumination light, for example. An excellent fluorescenceobservation image can not be obtained due to the influence of theinterference fringe.

[0015] Additionally, in the microscope disclosed in Jpn. Pat. Appln.KOKAI Publication No. 09-159922, the incident-light fluorescenceillumination may be turned on at the time of adjusting the incidenceangle from the glass to the sample when performing the fluorescenceobservation by the total internal fluorescence illumination in somecases. In this case, the mirror must be freshly moved to a position ofthe total internal reflection illumination. However, since the sample onthe glass surface is irradiated with the incident-light illuminationwith the strong intensity as the exciting light during this movement,the entire sample may lose its color.

[0016] Further, although the micrometer or the like is used to move themirror in the range from the incident-light fluorescence illumination tothe total internal reflection illumination, since the micrometer has asmall quantity of movement per one rotation of a rotation operationportion, the number of times of rotation increases when switching fromthe incident-light illumination to the total internal reflectionillumination. Therefore, a lot of trouble is taken until this switching,thereby greatly reducing the operability of the fluorescenceobservation. Furthermore, this means that the entire sample may possiblylose its color during this switching when trying to switch from theincident-light fluorescence illumination to the total internalreflection illumination while irradiating the sample with theillumination exciting light.

BRIEF SUMMARY OF THE INVENTION

[0017] A total internal reflection illumination apparatus according tothe first aspect of the present invention apparatus applied to amicroscope which illuminates a sample through an objective having anumerical aperture enabling total internal reflection illumination, ischaracterized by comprising: a first total internal reflection mirrorwhich is arranged in the vicinity of an outermost peripheral part of anobservation optical path of the microscope to reflect an incidentillumination light in a direction of the objective; a second totalinternal reflection mirror which is arranged at a symmetrical positionwith the first total internal reflection mirror to sandwich anobservation optical axis and reflects return light reflected on asurface of the sample in a direction different from the illuminationoptical path; and a return light dimming part configured to dim thereturn light reflected by the second total internal reflection mirror.

[0018] A microscope according to the second aspect of the presentinvention is characterized by comprising: a light source which emitspredetermined light; a total internal reflection illumination apparatusaccording to above-mentioned total internal reflection illuminationapparatus, which irradiates a sample with the light from the lightsource through an objective; and an image pickup device which images thelight from the sample as an image.

[0019] A microscope according to the third aspect of the presentinvention is characterized by comprising: a changing part configured tochange an incidence angle of illumination light which is emitted onto asample from a light source through an objective and enables switchingbetween total internal reflection illumination and approximate totalinternal reflection illumination; and a restricting part configured torestrict the incidence angle of the illumination light to the samplethrough the objective to a range where total internal reflectionillumination and approximate total internal reflection illumination canbe obtained.

[0020] Additional objects and advantages of the invention will be setforth in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention may be realized and obtained bymeans of the instrumentalities and combinations particularly pointed outhereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0021] The accompanying drawings, which are incorporated in andconstitute a part of the specification, illustrate presently preferredembodiments of the invention, and together with the general descriptiongiven above and the detailed description of the preferred embodimentsgiven below, serve to explain the principles of the invention.

[0022]FIG. 1 is a schematic structural view for illustrating a scheme ofa microscope according to the present invention;

[0023]FIG. 2 is a schematic structural view for illustrating the regularfluorescence incident-light illumination of the microscope according tothe present invention;

[0024]FIG. 3 is a view showing a schematic structure of a firstembodiment according to the present invention;

[0025]FIG. 4 is a view showing a schematic structure of a slider mainbody used in the first embodiment;

[0026]FIG. 5 is an overview diagram when a total internal reflectionillumination apparatus according to the first embodiment is applied toan inverted microscope;

[0027]FIG. 6 is an overview diagram when the total internal reflectionillumination apparatus according to the first embodiment is applied toan microscope with upright frame;

[0028]FIG. 7 is a view showing a schematic structure of a secondembodiment according to the present invention;

[0029]FIG. 8 is a view showing a schematic structure of a slider mainbody used in a third embodiment according to the present invention;

[0030]FIG. 9 is a view showing a schematic structure of a slider mainbody used in the third embodiment;

[0031]FIG. 10 shows a modification of the third embodiment;

[0032]FIG. 11 is a modification of the first embodiment to the thirdembodiment;

[0033]FIGS. 12A to 12C are views showing a schematic structure of afourth embodiment according to the present invention;

[0034]FIGS. 13A and 13B are views showing a schematic structure of amodification of the fourth embodiment according to the presentinvention;

[0035]FIG. 14 is a view showing a schematic structure of a fifthembodiment according to the present invention; and

[0036]FIGS. 15A to 15C show the case where the present invention isapplied to the microscope with upright frame.

DETAILED DESCRIPTION OF THE INVENTION

[0037] Embodiments according to the present invention will now bedescribed hereinafter with reference to the accompanying drawings.

[0038]FIG. 1 is a view for illustrating a scheme of total internalreflection fluorescence observation using a microscope. FIG. 1 shows anexample where an inverted microscope is used and observation is carriedout by an objective 7 arranged below a sample 2.

[0039] As shown in FIG. 1, a cover glass 3 is arranged on the lower sideof the sample 2. The objective 7 is provided below the cover glass 3through an immersion oil 5.

[0040] A mirror unit turret 9 which holds two or more fluorescencemirror units 10 a and 10 b and is capable of rotating (or linearlymoving (only the rotational operation will be described thereafter) isarranged on an optical axis 8 of the objective 7. The fluorescencemirror units 10 a and 10 b corresponding to the total internalreflection illumination or the incident-light fluorescence illuminationare selectively switched on the optical axis 8 by the rotationaloperation by a rotary shaft 14 of the mirror unit turret 9. In FIG. 1,the fluorescence mirror unit 10 a corresponding to the total internalreflection illumination is switched on the optical axis 8. Ahigh-reflection mirror 24 is arranged in the incidence light path of thefluorescence mirror units 10 a and 10 b. The high-reflection mirror 24is fixed to a mirror holding portion 22 by, e.g., an adhesive. Adovetail portion 22 a is provided to the mirror holding portion 22. Thedovetail portion 22 a is held in a dovetail groove portion 20 providedin an incident-light projection tube 17 so as to be capable of moving ina direction vertical to a page space in FIG. 1. The high-reflectionmirror 24 can be moved in the direction vertical to the page space bymoving in or out an operation knob 23 along the dovetail groove portion20. In this case, as shown in FIG. 1, when the high-reflection mirror 24is on the optical axis 19 of the incident-light projection tube 17, thelight from a laser beam source 41 is reflected to the fluorescencemirror units 10 a and 10 b side.

[0041] The laser beam from the laser beam source 41 is led from anoptical fiber incidence portion 40, and then outgoes from an opticalfiber outgoing portion 38. The outgoing light 21 a from the opticalfiber outgoing portion 38 is converted into parallel light 21 b by acollimator lens 29 of a fiber projection tube 28. The parallel light 21b is reflected by the high-reflection mirror 24, then condensed by acondenser lens 18, and led to the fluorescence mirror unit 10 a. Thefluorescence mirror unit 10 a includes a dichroic mirror 11 a and anabsorption filter 12 a. The light condensed by the condenser lens 18 isreflected by the dichroic mirror 11 a and focalized at a rear side focalposition 6 of the objective 7. The outgoing light from the end of theobjective 7 enters a sample 2 from the cover glass 3. Here, evanescentlight 4 which permeates in a range of several hundred nm from theinterface with the cover glass 3 can be generated on the sample (lowrefractive index side) 2 by shifting the optical axis 31 of the outgoinglight 21 a from the optical fiber outgoing portion 38 in a directionvertical to the optical axis 30 of the fiber projection tube 28 in sucha manner that an incidence angle of the incident light which is emittedfrom the end of the objective 7 and enters the sample (low refractiveindex side) 2 from the cover glass (high refractive index side) 3becomes larger than a critical angle.

[0042] A fluorescent material in the sample 2 which exists in thevicinity of the surface of the cover glass 3 where the evanescence light4 is generated is excited by the evanescence light 4 as the excitinglight and produces the fluorescence. The fluorescence passes through theobjective 7 and the dichroic mirror 11 a, and the disadvantageous lightin a wavelength band other than the fluorescence is eliminated therefromby the absorption filter 12 a. Then, the fluorescence is led to anobservation image formation system 15, divided into two light paths by ahalf mirror 60. One divided fluorescence is image-formed on ahigh-sensitivity camera (CCD or the like) 16. The other one istransmitted through the half mirror 60, reflected by the mirror 61, andobserved by an eyepiece 63. As a result, the fluorescent material in thesample 2 can be imaged or visually observed.

[0043] In case of performing the regular incident-light fluorescenceillumination, as shown in FIG. 2, the high-reflection mirror 24 isremoved from the optical axis 19 of the incident-light projection tube17, and the fluorescence mirror unit 10 a is switched to thefluorescence mirror unit 10 b for the incident-light fluorescenceillumination. The fluorescence mirror unit 10 b includes a dichroicmirror 11 b, an absorption filter 12 b and an exciting filter 13 b. Theexciting filter 13 b transmits therethrough only the exciting light inthe light beams from a mercury burner 26 of a mercury lamp house 25. Theexciting light transmitted through the exciting filter 13 b is reflectedby the dichroic mirror 11 b and enters the objective 7. The outgoinglight from the end of the objective 7 enters the sample 2 from the coverglass 3. The fluorescence from the fluorescent material in the sample 2is transmitted through the dichroic mirror 11 b, and the disadvantageouslight in the wavelength band other than the fluorescence is removedtherefrom by the absorption filter 12 b. Then, the fluorescence is ledto the observation image formation system 15, and divided into two lightpaths by the half mirror 60. One divided fluorescence is image-formed onthe high-sensitivity camera (CCD or the like) 16. The other one istransmitted through the half mirror 60, reflected by the mirror 61 andobserved by the eyepiece 63 through a relay optical system 62. As aresult, the fluorescent material can be imaged or visually observed.

[0044] Description will now be given as to embodiments of the totalinternal reflection illumination apparatus according to the presentinvention applied to the microscope having the above-describedstructure.

[0045] (First Embodiment)

[0046]FIG. 3 is a view showing a schematic structure of a microscope towhich the total internal reflection illumination apparatus according toa first embodiment of the present invention is applied. FIG. 3 is a viewshowing only the total internal reflection illumination apparatus andthe vicinity of the objective to which the total internal reflectionillumination apparatus is attached for the convenience's sake. It is tobe noted that any other structure is basically the same as theabove-described structure, thereby omitting illustration andexplanation. FIG. 4 is a view showing a schematic structure of a slidermain body used in the first embodiment.

[0047] In FIG. 3, an objective revolver holding base 71 is attached to anon-illustrated microscope main body.

[0048] A fixing portion 72 a of the objective revolver 72 is held to theobjective revolver holder base 71. A rotation portion 72 b is rotatablyprovided to the fixing portion 72 a of the objective revolver 72. Aplurality of (only one in the drawing) objectives 7 are screwed andfixed to the rotation portion 72 b. By rotating the rotation portion 72b, a desired objective 7 can be moved onto an observation optical axisO. In this case, circular hole portions 71 a and 72al having at thecenter thereof the observation optical axis O running through the centerof the objective 7 are provided to the objective revolver holding base71 and the fixing portion 72 a of the objective revolver 72.

[0049] The objective 7 has the numerical aperture enabling the totalinternal reflection illumination. An objective of the spotlight 7 a isprovided at the end of the objective 7. The objective of the spotlight 7a is arranged through the immersion oil 5 at a position opposed to thecover glass 3 having the specimen 2 mounted thereon.

[0050] A concave portion 71 b is formed on the top face of the objectiverevolver holding base 71. A box-like slider main body 80 as a supportmember is provided to the concave portion 71 b so as to be capable ofbeing inserted or removed along the surface orthogonal to theobservation optical axis O. Hole portions 6 a and 76 b through which theobservation optical path a runs are formed to the slider main body 80 onthe upper and lower surfaces thereof positioned on the observationoptical axis O while being attached to the concave portion 71 b of theobjective revolver holding base 71.

[0051] An optical fiber outgoing portion 38 is fixed to the side surfaceof the slider main body 80.

[0052] A convex lens 81 is arranged in the slider main body 80 at aposition opposed to the optical fiber outgoing portion 38. A first totalinternal reflection mirror 82 is arranged on the light path of the lightwhich is transmitted through a convex lens 81 and converged.

[0053] The convex lens 81 converts the scattered light beam emitted fromthe optical fiber 39 into the convergent light beam. The convex lens 81has such a focal distance as that the light reflected in the directionof the objective 7 by the first total internal reflection mirror 82 isfocalized at the rear side focal position 7 b of the objective 7.

[0054] Furthermore, the first total internal reflection mirror 82 isarranged in the vicinity of the outermost peripheral part of theobservation optical path a and reflects the convergent light beamtransmitted through the convex lens 81 in the direction of the objective7. The reflected light moves along the observation optical axis O in thevicinity of the outermost peripheral part of the inner space of theobjective 7.

[0055] A second total internal reflection mirror 83 is arranged at aposition symmetrical with respect to the first total internal reflectionmirror 82 so as to sandwich the observation optical axis O. The secondtotal internal reflection mirror 83 reflects in the direction differentfrom the observation optical path a the return light reflected on thesurface of the specimen 2 and returned along the observation opticalaxis O in the vicinity of the outermost peripheral part of the innerspace of the objective 7.

[0056] A light trap 85 as return light dimming part is arranged in thereflection direction of the second total internal reflection mirror 83.The light trap 85 dims the return light reflected by the second totalinternal reflection mirror 83.

[0057] As shown in FIG. 4, a first guide shaft 821 is provided to thefirst total internal reflection mirror 82. The first guide shaft 821 hasa linear portion 821 a and a bent portion 821 b obtained by the endportion of the linear portion 821 a substantially at right angles. Thefirst total internal reflection mirror 82 is attached to the bentportion 821 b. Moreover, the linear portion 821 a of the first guideshaft 821 is supposed by a pair of guides 822 a and 822 b. By linearlymoving the linear portion 821 a along the guides 822 a and 822 b, thefirst total internal reflection mirror 82 can be moved toward or awayfrom the observation optical axis O.

[0058] A second guide shaft 831 arranged in parallel with the firstguide shaft 821 is provided to the second total internal reflectionmirror 83. The second guide shaft 831 has a linear portion 831 a and abent portion 831 b obtained by bending the end portion of the linearportion 831 a substantially at right angles. The second total internalreflection mirror 83 is attached to the bent portion 831 b. In addition,the linear portion 831 a of the second guide shaft 831 is supported by apair of guides 832 a and 832 b. By linearly moving the linear portion831 a along the guides 832 a and 832 b, the second total internalreflection mirror 83 can be moved toward or away from the observationoptical axis O.

[0059] A link 84 is provided between the first guide shaft 821 and thesecond guide shaft 831. The intermediate portion of the link 84 isrotatably supposed on the bottom surface of the slider main body 80. Theboth ends of the link 84 are rotatably attached to the linear portion821 a of the first guide shaft 821 and the linear portion 831 a of thesecond guide shaft 831, respectively. As a result, the second guideshaft 831 moves in the direction opposed to the movement direction ofthe first guide shaft 821 with respect to movement of the first guideshaft 821.

[0060] Additionally, a spring 84 a is provided to the link 84 at aposition between the link 84 and the bottom surface of the slider mainbody 80. This spring 84 a constantly gives the link 84 a running torquein the counterclockwise direction.

[0061] An operation shaft 823 pierces a screw portion 823 a and isprovided to the side surface of the slider main body 80. The operationshaft 823 is in contact with the end portion of the first guide shaft821. A quantity of screwing of the screw portion 823 a is adjusted byoperation of a knob 823 b, and the first guide shaft 821 is linearlymoved. As a result, the first total internal reflection mirror 82 andthe second total internal reflection mirror 83 can be moved close to oraway from the observation optical axis O while maintaining thepositional relationship of symmetry so as to sandwich the observationoptical axis O.

[0062] The operation of the first embodiment having the above-describedstructure will now be described.

[0063] When the light is emitted from the optical fiber outgoing portion38, it becomes the scattered light beam and enters the convex lens 81.The scattered light beam which has entered the convex lens 81 isconverted into the convergent light beam, then reflected in thedirection of the objective 7 by the first total internal reflectionmirror 82, and moves along the observation optical axis O in thevicinity of the outermost part of the inner space of the objective 7.Then, the light beam is focalized at the rear side focal position 7 b ofthe objective 7, again becomes the scattered light beam, and istransmitted through the objective of the spotlight 7 a. Thereafter, thelight beam becomes the parallel light beam inclined with respect to theobservation optical axis O and outgoes from the objective 7. Then, it istransmitted through the immersion oil 5 and the cover glass 3, and thespecimen 2 is irradiated with this light.

[0064] Here, when the operation shaft 823 is rotated by the operation ofthe knob 823 b, the first guide shaft 821 and the second guide shaft 831linearly move in the opposite directions in accordance with a quantityof screwing of the screw portion 823 a. The first total internalreflection mirror 82 ad the second total internal reflection mirror 83are subjected to positional adjustment in the direction to move closerto or away from the observation optical axis O while maintaining thepositional relationship of symmetry with respect to the observationoptical axis O. As a result, the distance of the light beam which movesin the inner space of the objective 7 from the observation optical axisO varies, and the inclination angle of the parallel light beam outgoingfrom the objective 7 changes. That is, the total internal reflectionillumination can be obtained by changing the inclination angle of theparallel light beam outgoing from the objective 7 by rotating the knob823 b of the operation shaft 823 and setting this inclination angleequal to or above a critical angle.

[0065] On the other hand, the light beam totally reflected on thesurface of the specimen 2 again enters the objective 7 as the returnlight, is focalized at the rear side focal position 7 b of the objective7, all reflected by the second total internal reflection mirror 83, ledto the light trap 85 and terminated.

[0066] In this state, when the fluorescence is emitted from the specimen2, this florescence moves in the observation optical path a along theobservation optical axis O as an observation light beam O′. Theobservation light beam O′ at this moment is led to the observation sidewithout crossing the illumination light beam reflected by the firsttotal internal reflection mirror 82 or the return light beam whichenters the second total internal reflection mirror 83. Additionally, theillumination light beam with which the specimen 2 is irradiated and thereturn light beam after total internal reflection do not cross eachother on the observation optical path a and they are eliminated from theobservation optical path a.

[0067] Therefore, according to the above-described structure, theobservation light beam O′ of the fluorescence emitted from the specimen2 can be set so as not to cross the illumination light beam reflected bythe first total internal reflection mirror 82 or the return light beamentering the second total internal reflection mirror 83 on theobservation optical path a. Accordingly, the self-fluorescence generatedon the illumination light beam or the return light beam can be preventedfrom entering the observation light beam O′, thereby greatly suppressingdeterioration of the fluorescence observation image. Further, since theillumination light beam with which the specimen 2 is irradiated and thereturn light beam after total internal reflection do not cross eachother, the interference fringe generated due to crossing of the laserbeams can be prevented, thereby constantly assuring the excellentfluorescence observation image.

[0068] It is to be noted that the interlocking mechanism constituted bythe first guide shaft 821, the second guide shaft 831 and the link 84are used as a movement part of the first total internal reflectionmirror 82 and the second total internal reflection mirror 83 is used inthe first embodiment but the present invention can be embodied by usingany other moving parts, e.g., using an electric motor in place of theinterlocking mechanism. Further, the convex lens 81 is of the fixedtype, but it may be capable of moving along the illumination light beam,thereby enabling the precise optical adjustment.

[0069]FIGS. 5 and 6 show the schematic structures obtained when thetotal internal reflection illumination apparatus according to the firstembodiment having the above-described structure is applied to invertedmicroscope and microscope with upright frame. FIG. 5 is a view when thetotal internal reflection illumination apparatus is applied to theinverted microscope, and FIG. 6 is a view when the total internalreflection illumination apparatus is applied to the microscope withupright frame. Incidentally, in FIGS. 5 and 6, like reference numeralsdenote parts equal to those in FIGS. 1 to 4, thereby omitting thedetailed description.

[0070] As shown in FIG. 5, in the inverted microscope, a light sourcefor incident-light illumination is arranged above a stand 100, andregular observation of a sample is carried out with the eyepiece 63 byilluminating the sample 2 on the cover glass 3 through an incident-lightoptical system 96. In this case, the sample 2 is not illuminated withthe laser beam from the laser beam source 41 using the slider main body80. Further, when performing observation by the total internalreflection illumination using the total internal reflection illuminationapparatus, the fluorescence from the sample 2 is observed by performingthe total internal reflection illumination to the sample 2 with thelaser beam from the laser beam source 41 (or image pickup is carried outby a non-illustrated image pickup device).

[0071] In case of FIG. 6, the illumination from a non-illustrated lightsource is emitted toward the upper direction from the lower part of thestand 100′ along the optical axis of the objective 7. Any otherstructure is substantially the same with as of FIG. 5, thereby omittingthe description.

[0072] (Second Embodiment)

[0073]FIG. 7 is a view showing a schematic structure of a microscope towhich a total internal reflection illumination apparatus according to asecond embodiment of the present invention is applied. In FIG. 7, likereference numerals denote parts equal to those in FIG. 3, therebyomitting the detailed explanation.

[0074] In the second embodiment, the convex lens 851 is arranged on thereflected light path of the second total internal reflection mirror 83.The convex lens 851 is used to again converge the return light subjectedto total internal reflection by the second total internal reflectionmirror 83. An incidence end 852 a of the optical fiber 852 is positionedat a focal position of the return light obtained by the convex lens 851.In this case, the incidence end 852 a of the optical fiber 852 has asufficiently large core diameter in such a manner that the light can beassuredly led into the fiber even if the focal position of the returnlight slightly deviates. Furthermore, the light trap 853 is connected toan outgoing end 22 b of the optical fiber 852. This light trap 853 isset outside the microscope.

[0075] Description will be given as to the operation of the secondembodiment having the above-described structure.

[0076] The light beam totally reflected by the surface of the specimen 2again enters the objective 7 as the return light, and is focalized atthe rear side focal position 7 b of the objective 7. Thereafter, thereturn light is all reflected by the second total internal reflectionmirror 83 and enters the convex lens 851. Then, the return light istransmitted through the convex lens 851, again becomes the convergentlight beam, enters the incidence end 852 a of the optical fiber 852, andis terminated at the light trap 853 provided outside the microscope.

[0077] By doing so, the advantages like those in the first embodimentcan be obtained. Moreover, since the return light beam reflected by thesecond total internal reflection mirror 83 is positively taken out tothe outside of the microscope by using the optical fiber, thepossibility of permeation of the scattered light of the return lightbeam into the observation optical path a can be greatly reduced, therebyassuring the further excellent fluorescence observation image.

[0078] (Third Embodiment)

[0079]FIGS. 8 and 9 are views showing a schematic structure of a slidermain body according to a third embodiment of the present invention. InFIGS. 8 and 9, like reference numerals denote parts equal to those inFIG. 4, thereby omitting the detailed explanation.

[0080] In FIGS. 8 and 9, a pair of click grooves 825 and 826 providedwith a predetermined gap therebetween along the side surface which is incontact with the concave portion 71 b to the slider main body 80disposed so as to be capable of being inserted to or removed from theconcave portion 71 b of the objective revolver holding base 71.Moreover, a ball plunger 827 is provided on the side surface of theobjective revolver holding base 71 on the concave portion 71 b side,with which the slider main body 80 is in contact.

[0081] The ball plunger 827 constantly presses the side surface of theslider main body 80, and positions the slider main body 80 when fittedin the click grooves 825 and 826. When the slider main body 80 isinserted to reach the observation optical axis O as shown in FIG. 8, theball plunger 827 is fitted in the click groove 825. When the slider mainbody 80 is moved away from the observation optical axis O as shown inFIG. 9, the ball plunger 827 is fitted in the click groove 826.

[0082] A micro switch 86 is arranged to the concave portion 71 b of theobjective revolver holding base 71 at a position where it is pressed bythe end of the slider main body 80. The micro switch 86 is pressed andbecomes conductive when the slider main body 80 is being inserted in theobservation optical axis O as shown in FIG. 8.

[0083] An interlocking terminal 41 a of a laser oscillator 41 isconnected to an output terminal of the micro switch 86 through a cable87. The laser oscillator 41 oscillates when interlocking is released byconduction of the micro switch 86, and generates the laser beam. Thelaser beam is led to the slider main body 80 through the optical fiber39.

[0084] The operation of the third embodiment having such a structurewill now be described.

[0085] As shown in FIG. 8, when the slider main body 80 is inserted ontothe observation optical axis O, the ball plunger 827 is fitted in theclick groove 825, and the slider main body 80 is held so as not toeasily move. When the micro switch 86 is pressed by the end of theslider main body 80 and becomes conductive in this state, theinterlocking of the laser oscillator 41 is released, and the laser beamis generated. The laser beam is led to the slider main body 80 throughthe optical fiber 39, and the operation like that described inconnection with the first embodiment can be obtained.

[0086] On the other hand, as shown in FIG. 9, when the slider main body80 is moved away from the observation optical axis O, the ball plunger827 is fitted in the click groove 826, and the slider main body 80 isheld so as not to easily move. In this state, since the micro switch 86is opened, the interlocking of the laser oscillator 41 connected to thecable 87 is actuated, thereby generating no laser beam.

[0087] Therefore, the advantages like those described in connection withthe first embodiment can be obtained by doing so. Furthermore, when theslider main body 80 is removed from the observation axis O, the laserbeam from the laser oscillator 41 can be automatically stopped surely.Thus, the scattered light by the reflection of the laser beam neverpermeates observation light axis O′ and excellence can be maintained.

[0088] It is to be noted that the cable 87 is directly connected to theinterlocking terminal 41 a of the laser oscillator 41 in the thirdembodiment, the similar advantages can be obtained by inserting anelectric shutter between the laser oscillator 41 and the optical fiber39, connecting the cable 87 to the electric shutter and using a part of,e.g., combining the opening/closing operation of the electric shutterwith the on/off operation of the micro switch 86.

[0089] Further, in the third embodiment, the micro switch 86 isconfigured to be pressed and conductive with the slider main body 80being inserted into the observation optical axis O, but the micro switch86 may be provided on the side surface of the inner wall as shown inFIG. 10. It is to be noted that the micro switch 86 may be of a contacttype or it may be a proximity switch or the like in this case.

[0090] In the first to third embodiments mentioned above, in the firstguide shaft 821 and the second guide shaft 831 which is arranged inparallel with the first guide shaft 821, there is no structure whichrestricts a quantity of movement of these shafts. Accordingly, as shownin FIG. 11, there may be provided a stopper which restricts a quantityof movement of the first guide shaft 821 and the second guide shaft 831.This stopper can prevent the reflection mirror from moving in theoptical axis direction in particular.

[0091] Although the micro switch 86 is arranged at a position where itis pressed by the end of the slider main body 80 in the thirdembodiment, the micro switch 86 may be provided at such a position asthat it is pressed by the side surface of the slider main body 80 asshown in FIG. 10. The micro switch 86 may be provided at any position aslong as it can detect insertion and removal of the slider main body 80in this way. Moreover, any kind of the micro switch 86, e.g., amechanical type, a proximity sensor or the like can be used.

[0092] In addition, in the first to third embodiments, the first guideshaft 821 and the second guide shaft 831 can be moved by the operationshaft 823 and the link 84, but a quantity of movement of the first guideshaft 821 and the second guide shaft 83 is restricted by providing astopper 88 so as to come into contact with one of the second guide shaft831 as shown in FIG. 11, and the first total internal reflection mirror82 and the second total internal reflection mirror 83 do not move closeto the optical axis more than necessary, thereby preventing damages tothese members.

[0093] (Fourth Embodiment)

[0094]FIGS. 12A to 12C are views showing a schematic structure of amicro scope to which a fourth embodiment according to the presentinvention is applied. In FIGS. 12A to 12C, like reference numeralsdenote parts equal to those in FIG. 1, thereby omitting the detaileddescription.

[0095] The incident-light projection tube 17 is fixed to the invertedmicroscope main body (not shown). This incident-light projection tube 17has a connection portion 17 relative to a fiber projection tube 28, anda connection portion 17 b relative to a mercury lamp house 25 whichholds the mercury burner 26. These members are respectively connected insuch a manner that an optical axis 30 of the fiber projection tube 28becomes orthogonal to an optical axis 19 of the incident-lightprojection tube 17 and an optical axis 27 of the mercury lamp house 25coincides with the optical axis 19 of the incident-light projectiontube.

[0096] In the incident-light projection tube 17, the high-reflectionmirror 24 is fixed to the mirror holding portion 22 by an adhesive orthe like so as to reflect the parallel beam 21 b of the fiber projectiontube 28 on the optical axis 19 of the incident-light projection tube 17.A dovetail portion 22 a is provided to the mirror holding portion 22.The dovetail portion 22 a is held in the dovetail groove portion 20provided to the incident-light projection tube 17 so as to be capable ofmoving in the direction vertical to the page space, and thehigh-reflection mirror 24 can be moved in the direction vertical to thepage space by moving in or out the operation knob 23 in the directionvertical to the page space from the outside of the incident-lightprojection tube 17. Moreover, the mirror holding portion 22 has a lightshielding portion 22 b provided on the side surface thereof on themercury lamp house 25 side.

[0097] The fiber projection tube 28 is constituted by a collimator lens29 and a fiber lead-in portion 32. The fiber lead-in portion 32 isconnected to the end of the fiber projection tube 28 on the sideopposite to the incident-light projection tube 17.

[0098] On the other hand, the outgoing light from the laser beam source41 enters the optical fiber 39 from the optical fiber incidence portion40, and the outgoing light 21 a outgoes from the optical fiber outgoingportion 38. The optical fiber outgoing portion 38 is fixed to themovement portion 37 by a screw or the like (not shown). An outer sidesurface portion 37 a of the movement portion 37 is fitted with an innerside surface portion 32 a 1 of the fiber lead-in portion 32 and can movein the horizontal direction of the page space 54.

[0099] Here, a screw hole 32 a having a central line 36 in parallel withthe horizontal direction of the page space 54 of the movement portion 37and a fitting hole 32 b are formed to the fiber lead-in portion 32, anda lid cylinder 33 having a screw portion is engaged with the screw hole32 a, and an adapter 35 is fitted in the fitting hole 32 b.

[0100] In addition, a compression coil spring 34, which is compressed tobe shorter than a natural length, as an elastic body is sandwichedbetween the lid cylinder 33 and the adapter 35, and the adapter 35 comesinto contact with the outer side surface portion 37 a of the movementportion 37. On the other hand, a slant surface contact portion 37 b isprovided on the opposite side to the outer side surface portion 37 a ofthe movement portion 37.

[0101] A micrometer holding portion 42 is fixed to the fiber lead-inportion 32 by a screw or the like (not shown). The micrometer main body44 is held to the micrometer holding portion 42 by a screw or the like(not shown).

[0102] The micrometer main body 44 has a knob 46 engage with therotation portion 44 a by a screw or the like (not shown). Additionally,a screw hole 32 a and a cylindrical hole 32 c which has a central lineorthogonal to the central line of the fitting hole 32 b are provided tothe fiber lead-in portion 32, and a capsule adapter 43 is arranged beingfitted in the cylindrical hole 32 c and sandwiched between the slantsurface contact portion 37 b and the end portion 44 b of the rotationportion 44 a of the micrometer main body 44.

[0103] As a result, the knob 46 of the micrometer main body 44 isrotated, the slant surface contact portion 37 b of the movement portion37 is pressed by the capsule adapter 43 of the end portion 44 b of therotation portion 44 a, and the movement portion 37 is moved against thepressing force of the compression coil spring 34. Consequently, theoptical axis 31 of the outgoing light 21 a from the optical fiberoutgoing portion 38 can be shifted from the optical axis 30 of the fiberprojection tube 28 (to the left side in the drawing) so as to be capableof adjusting an incidence angle of the incident light which outgoes fromthe end of the objective 7 and enters the sample (low refractive indexside) 2 from the cover glass (high refractive index side) 3.

[0104] An opening portion 45 a of a notch stopper 45 which has theopening portion 45 a and an U-shaped notch portion 45 c is fitted to thefixing portion 44 c of the micrometer main body 44 as restricting partas shown in FIG. 12C. Additionally, a screw 47 which adjusts a gap ofthe U-shaped notch portion 45 c is provided.

[0105] The operation of the fourth embodiment having such a structurewill now be described.

[0106] The adapter 35 is in the state that the outer side surfaceportion 37 a of the movement portion 37 is pressed by the compressioncoil spring 34. On the other hand, the end portion 44 b can be moved inthe vertical direction of the page space 55 by rotating the knob 46 ofthe micrometer main body 44, and the movement portion 37 can be moved inthe horizontal direction of the page space 54 through the capsuleadapter 43. As a result, the optical axis 31 of the outgoing light 21 afrom the optical fiber outgoing portion 38 can be adjusted in thevertical direction relative to the optical axis 30 of the fiberprojection tube 28 while maintaining the horizontal state with respectto the optical axis 30 of the fiber projection tube 28.

[0107] At the same time, an angle of the incident light which is emittedfrom the end of the objective 7 and enters the sample 2 from the coverglass 3 can be also adjusted. Here, a position of the movement portion37 is adjusted by rotating the knob 46 in such a manner that theincidence angle from the cover glass 3 to the sample 2 becomes slightlylarger than the critical angle, and the side surface portion 45 d of thenotch stopper 45 is pressed against a contact portion 44 aa of therotation portion 44 a of the micrometer main body 44 at this position.Further, a gap of the notch portion 45 c of the notch stopper 45 isconstricted by a screw 47, and the fixing portion 44 c of the micrometermain body 44 is shut in by the opening portion 45 a, thereby positioningand fixing the notch stopper 45 with respect to the micrometer main body44.

[0108] Therefore, the movement portion 37 can not thereafter move theoptical fiber outgoing portion 38 to the optical axis 30 side of thefiber projection tube 28 by restriction of the notch stopper 45, andmovement is limited to that only in a range of the total internalreflection illumination that the incidence angle from the cover glass 3to the sample 2 is larger than the critical angle.

[0109] On the other hand, although the light beam (not shown) from themercury lamp house 25 is prevented by the light shielding portion 22 bof the mirror holding portion 22, light beam (not shown) from themercury burner 26 can be led to the incident-light projection tube 17 bydrawing the operation knob 23 toward the front side in the verticaldirection of the page space and removing the mirror holding portion 22from the light path. At this moment, by rotating the mirror unit turret9 around the rotary shaft 14, the regular incident-light fluorescenceillumination observation can be enabled by arranging the fluorescencemirror unit 10 b including the exciting filter 13 b, the dichroic mirror11 b and the absorption filter 12 on the optical axis.

[0110] Therefore, according to the fourth embodiment, when adjusting theincidence angle from the cover glass 3 to the sample 2 by the notchstopper 45 fixed to the micrometer main body, this incidence angle isrestricted in a range where it becomes larger than the critical angle,and hence the fluorescence observation can be performed by only thetotal internal reflection illumination. Therefore, it is possible toprevent color degradation of the entire sample due to the strong lightof the incident-light fluorescence illumination, thereby obtaining thestable fluorescence observation by the total internal reflectionillumination. Furthermore, since the incidence angle from the coverglass 3 to the sample 2 can be constantly adjusted in the range of thetotal internal reflection illumination, the operability can be improved.Moreover, the total internal reflection illumination and theincident-light fluorescence illumination can be rapidly switched byinsertion and removal of the high-reflection mirror 24, and hence afactor of color degradation of the sample can be avoided when switchingfrom the incident-light fluorescence illumination to the total internalreflection illumination (or vice versa) while keeping illumination ofthe sample with the illumination light.

[0111] It is to be noted that the inverted microscope has been describedin the fourth embodiment but the similar advantages can be obtained whenthe present invention is applied to the microscope with upright frame.(Modification of Fourth Embodiment) A modification of the fourthembodiment will now be described. FIGS. 13A and 13B illustrate amodification of the fourth embodiment, and like reference numeralsdenote parts equal to those in FIGS. 12A to 12C, thereby omitting thedetailed description.

[0112] In FIGS. 13A and 13B, a slide opening portion 28 a is provided tothe fiber projection tube 28 on the rear side (incident-light projectiontube 17 side) of the collimator lens 29. A slider 48 having an opening48 a and an opening 48 b is provided to the slide opening portion 28 aso as to be capable of moving in the horizontal direction of the pagespace 54. The slider 48 has a diffused plate 49 fixed to the opening 48a by a ring screw (not shown).

[0113] On the other hand, the mirror holding portion 22 of thehigh-reflection mirror 24 is directly fixed to a fixing portion 50 ofthe incident-light projection tube 17 by a screw or the like (notshown).

[0114] With such a structure, when the opening 48 b of the slider 48 isarranged on the optical axis 30 of the fiber projection tube 28 with thetotal internal reflection illumination being set as described inconnection with the fourth embodiment, this state is maintained, but theparallel light 21 b is diffused and the illumination is switched to theincident-light fluorescence illumination when the slider 48 is moved andthe opening 48 a having the diffused plate 49 is arranged on the opticalaxis 30.

[0115] By doing so, the advantages like those in the fourth embodimentcan be obtained, and switching between the total internal reflectionillumination and the incident-light fluorescence illumination can berealized without using the mercury lamp house 25.

[0116] Moreover, in the fourth embodiment, the light stopper 85 whichintroduces return light of the total reflected laser beam to an outsideof the observation optical path is provided between the objective 7 andthe fluorescence mirror unit 10 a like the first to third embodiments.As a result, the unnecessary light can be prevented from entering theobservation optical path, thereby obtaining a further excellent image.

[0117] It is to be noted that the high-reflection mirror 24 can beomitted by providing the fiber projection tube 28 coaxially with theincident-light projection tube 17 in the modification of the fourthembodiment, thereby obtaining the further inexpensive structure.

[0118] (Fifth Embodiment)

[0119] The fifth embodiment according to the present invention will nowbe described. FIG. 14 illustrates the fifth embodiment, and likereference numerals denote parts equal to those in FIG. 12A.

[0120] In FIG. 14, a dovetail portion 51 is provided to the mirrorholding portion 22 of the high-reflection mirror 24, and this dovetailportion 51 is held so as to be capable of moving in the horizontaldirection of the page space 54 in a dovetail groove portion 52 providedto the incident-light projection tube 17.

[0121] In the mirror holding portion 22, a contact portion 51 a of thedovetail portion 51 is constantly being pressed by the compression coilspring 34 through the adapter 35. On the other hand, a micrometerholding portion 53 is provided on the side surface of the incident-lightprojection tube 17 on the side opposite to a position where the adapter35 is arranged, and the micrometer main body 44 is provided to themicrometer holding portion 53. In this case, the micrometer main body 44brings the end portion 44 b of the rotation portion 44 a rotated byrotation of the knob 46 into contact with the contact portion 51 b ofthe dovetail portion 51.

[0122] By rotating the knob 46 of the micrometer main body 44 in thisstate, the mirror holding portion 22 can be moved along the reflectedlight path of the high-reflection mirror 24 by the dovetail portion 51,and a reflection position of the parallel light 21 b on thehigh-reflection mirror 24 can be adjusted.

[0123] As a result, the reflected light 21 c obtained from the parallellight 21 b on the high-reflection mirror 24 can be shifted from theoptical axis 19, and an angle of the incident light which outgoes fromthe objective 7 and enters the sample 2 from the cover glass 3 can beadjusted.

[0124] Here, a position of the mirror holding portion 22 is alsoadjusted by rotating the knob 46 in such a manner that the incidenceangle from the cover glass 3 to the sample 2 becomes slightly largerthan the critical angle, the notch stopper 45 is brought into contactwith the rotation portion 44 a of the micrometer main body 44 at thisposition, and the notch stopper 45 is fastened, thereby performingpositioning and fixing with respect to the micrometer main body 44.

[0125] As a result, the mirror holding portion 22 can not thereaftermove the reflected light 21 c from the high-reflection mirror 24 to theoptical axis 19 side by the restriction of the notch stopper 45, andmovement is restricted to that only in the range of the total internalreflection illumination that the incidence angle from the cover glass 3to the sample 2 is larger than the critical angle.

[0126] In this case, like the modification of the fourth embodiment, theparallel light 21 b is diffused by providing the slider 48 and arrangingthe opening 48 a having the diffused plate 49 on the optical axis 30,thereby obtaining the incident-light fluorescence illumination utilizingthe laser beam source 41.

[0127] In the fifth embodiment, although the mirror holding portion 22is moved along the reflected light path of the high-reflection mirror24, the similar advantages can be obtained by moving it along theincident-light path of the high-reflection mirror 24.

[0128] It is to be noted that the micrometer main body 44 and the notchstopper 45 which restricts movement of the rotation portion 44 a of themicrometer main body 44 are used in the fifth embodiment. However,instead of not using these members, a light shielding plate having aslit hole shifted to the right or left side from the center of theoptical axis 30 of the fiber projection tube 28 may be arranged in frontof the optical fiber outgoing portion 38, and the optical axis of theoutgoing light emitted from the optical fiber outgoing portion 38 may bemoved to the optical axis 30 side of the fiber projection tube 28. Evenin this case, by preventing the light on the optical axis center side ofthe outgoing light by using the light shielding plate, switching to theincident-light illumination can be avoided, and movement can berestricted to that only in the range of the total internal reflectionillumination. In this case, switching between the total internalreflection illumination and the incident-light fluorescence illuminationutilizing the laser beam source 41 can be performed by moving thehigh-reflection mirror 24 so as to be inserted into or removed from thelight path as with the fourth embodiment.

[0129] Description will now be given as to a method for realizing thetotal internal reflection illumination. In the confirmation method whenthe incidence angle of the illumination light from the cover glass 3 tothe sample 2 exceeds the critical angle, the eyepiece (not shown)attached to the body tube (not shown) is replaced with a CT (centeringtelescope) (not shown), and a lens group (not shown) in the vicinity ofthe rear side focal position 6 of the objective 7 is watched by usingthis CT. The self-fluorescence of the lens group is generated by thelight obtained by transmission of the illumination light through thelens group (not shown) of the objective 7, and a bright spot can beobserved by the lens group in the vicinity of the rear side focalposition 6 at which the light is condensed.

[0130] When the incidence angle from the cover glass 3 to the sample 2is smaller than the critical angle, only one bright spot on theincidence angle side of the illumination light is observed at the rearside focal position 6 of the objective 7, but two bright spots which aresymmetric so as to sandwich the optical axis can be observed in thevicinity of the inner side of the outer periphery of the rear side focalposition 6 of the objective 7 when the incidence angle is larger thanthe critical angle. The second bright spot is obtained because theillumination light totally reflected on the interface between the coverglass 3 and the sample 2 returns to the objective 7 from the end of theobjective 7.

[0131] By gradually changing from the state that the incidence angle ofthe illumination light from the cover glass 3 to the sample 2 is smallerthan the critical angle to the state that it is larger than the criticalangle, one bright spot first gradually moves in the outer peripheraldirection from the central side of the optical axis of the objective.Then, when the incidence angle exceeds the critical angle, the secondbright point appears symmetrically so as to sandwich the optical axis ofthe objective, and the notch stopper 45 is fixed to the micrometer mainbody 44 when the second bright spot appears.

[0132] Description will now be given as to the definition of theapproximate total internal reflection illumination and effects andadvantages obtained when the present invention is applied to the fourthembodiment based on the fourth embodiment. Since the structure issimilar to that of the fourth embodiment, the explanation thereof isomitted. Further, in regard to the effects and the advantages, partsequal to those in the fourth embodiments are omitted, and descriptionwill be given as to only different parts.

[0133] The approximate total internal reflection illumination is firstdefined as follows. In the total internal reflection illumination, theevanescent light 4 is generated in the range of several hundred nm onthe sample 2 side which is the low refractive index medium side on theinterface between the cover glass 3 and the sample 2. By setting theincidence angle from the cover glass 3 to the sample 2 slightly smallerthan the critical angle, the refracted light from the cover glass 3 tothe sample 2 is emitted from the cover glass 3 along the vicinity on theinterface of the sample 2. In this illumination method, a range ofseveral nm in the sample 2 in the vicinity of the cover glass 3 can beilluminated. This is one type of dark field illumination, and thisillumination method is referred to as “approximate total internalreflection illumination” in this specification.

[0134] The effects of the approximate total internal reflectionillumination will now be described. Here, description will be given asto only the approximate total internal reflection illumination using thelaser beam source 41, and the explanation about the incident-lightfluorescence illumination using the mercury burner 26 as a light sourcewill be omitted. Like the fourth embodiment, the knob 46 of themicrometer 44 is first rotated, and illumination is switched to thetotal internal reflection illumination that the incidence angle from thecover glass 3 to the sample 2 is larger than the critical angle. Then,the knob 46 of the micrometer main body 44 is gradually rotated in thedirection that the incidence angle from the cover glass 3 to the sample2 becomes smaller, and the illumination light is adjusted to be theabove-described approximate total internal reflection illumination.

[0135] In this state, like the fourth embodiment, movement of theoptical fiber outgoing portion 38 is restricted to only the range of theapproximate total internal reflection illumination and the totalinternal reflection illumination by positioning and fixing the notchstopper 45 to the micrometer main body 44.

[0136] The advantages will now be described. The illumination range isonly the sample 2 in the range of several μm in the vicinity of the topface of the cover glass 3 even in case of the approximate total internalreflection illumination, and color degradation of the entire sample 2can be avoided like the fourth embodiment. Also, the illumination lightcan be moved in the direction away from the optical axis in order tochange to the total internal reflection illumination.

[0137] Besides the above advantages, an area which can not be observedwith the evanescent light 4 generated by the total internal reflectionillumination or the sample 2 which can not be observed because thegenerated fluorescence is too weak can be observed. Furthermore, thefluorescence is generated from the entire sample 2 in the incident-lightfluorescence illumination using the mercury burner 26 as a light source,whereas the unnecessary fluorescence can be eliminated and observationwith less background noise is enabled since the illumination range canbe restricted to the range of several μm in the vicinity of the coverglass 3 in the approximate total internal reflection illumination.

[0138] Although description has been give as to the inverted typemicroscope in the fourth and fifth embodiments mentioned above, thepresent invention can be of course applied to the erect type microscope.FIGS. 15A to 15C show the case where the present invention is applied tothe microscope with upright frame. In FIGS. 15A to 15C, since respectivestructures are the same as above, like reference numerals are given,thereby omitting the explanation.

[0139] The following inventions can be extracted from each of theforegoing embodiments. It is to be noted that each of the followinginventions may be appropriately combined and applied or it may beapplied independently.

[0140] A total internal reflection illumination apparatus according tothe first aspect of the present invention apparatus applied to amicroscope which illuminates a sample through an objective having anumerical aperture enabling total internal reflection illumination, ischaracterized by comprising: a first total internal reflection mirrorwhich is arranged in the vicinity of an outermost peripheral part of anobservation optical path of the microscope to reflect an incidentillumination light in a direction of the objective; a second totalinternal reflection mirror which is arranged at a symmetrical positionwith the first total internal reflection mirror to sandwich anobservation optical axis and reflects return light reflected on asurface of the sample in a direction different from the illuminationoptical path; and a return light dimming part configured to dim thereturn light reflected by the second total internal reflection mirror.

[0141] In the first aspect the flowing modes are preferable. Thefollowing modes may be applied independently or in combining them.

[0142] (1) The return light dimming part has an external set portionwhich is set outside the microscope.

[0143] (2) The return light dimming part is connected to the externalset portion through an optical fiber.

[0144] (3) At least a part of the total internal reflection illuminationapparatus is set to a support member which insertable or removable tothe observation optical path of the microscope.

[0145] (4) The first total internal reflection mirror and the secondtotal internal reflection mirror are arranged at positions where theyare symmetrical with each other to sandwich an optical axis of theobjective, and the first total internal reflection mirror and the secondtotal internal reflection mirror move in such a manner a distancebetween the optical axis of the objective and the first total internalreflection mirror and a distance between the optical axis of theobjective and the second total internal reflection mirror become equalto each other.

[0146] (5) One of the microscope and the support member has a switchingpart configured to turn on or turn off the illumination light incooperation with insertion or removal of the support member.

[0147] (6) A switching part configured to switch total internalreflection illumination and approximate total internal reflectionillumination by changing an incidence angle of the illumination lightwith which the sample is irradiated; and a restricting part configuredto restrict the incidence angle of the illumination light to the sampleto a range where total internal reflection illumination and approximatetotal internal reflection illumination can be obtained are furtherprovided.

[0148] A microscope according to the second aspect of the presentinvention is characterized by comprising: a light source which emitspredetermined light; a total internal reflection illumination apparatusaccording to above-mentioned total internal reflection illuminationapparatus, which irradiates a sample with the light from the lightsource through an objective; and an image pickup device which images thelight from the sample as an image.

[0149] In the second aspect the flowing modes are preferable. Thefollowing modes may be applied independently or in combining them.

[0150] (1) The light source has an optical fiber having an outgoing endprovided to be movable in a direction vertical to the optical axis, andthe restricting part restricts a movement range of the outgoing end ofthe optical fiber in the direction vertical to the optical axis to therange where total internal reflection illumination and approximate totalinternal reflection illumination can be obtained.

[0151] (2) An optical element which is insertably and removably arrangedto the light path between the light source and the objective anddiffuses the illumination light is further provided.

[0152] (3) A light path between the light source and the objective has areflection member arranged movably along a direction of the light path,and the restricting part restricts a movement range of the reflectionmember along the direction of the light path to the range where totalinternal reflection illumination and approximate total internalreflection illumination can be obtained.

[0153] (4) An optical element which is insertably and removably arrangedto the light path between the light source and the objective anddiffuses the illumination light is further provided.

[0154] A microscope according to the third aspect of the presentinvention is characterized by comprising: a changing part configured tochange an incidence angle of illumination light which is emitted onto asample from a light source through an objective and enables switchingbetween total internal reflection illumination and approximate totalinternal reflection illumination; and a restricting part configured torestrict the incidence angle of the illumination light to the samplethrough the objective to a range where total internal reflectionillumination and approximate total internal reflection illumination canbe obtained.

[0155] In the third aspect the flowing modes are preferable. Thefollowing modes may be applied independently or in combining them.

[0156] (1) A dimming part configured to dim light reflected by thesample is further provided.

[0157] (2) The light source has an optical fiber having an outgoing endprovided to be movable in a direction vertical to an optical axis, andthe restricting part restricts a movement range of the outgoing end ofthe optical fiber in the direction vertical to the optical axis to therange where total internal reflection illumination and approximate totalinternal reflection illumination can be obtained.

[0158] (3) An optical element which is insertably and removably arrangedthe light path between the light source and the objective and diffusesthe illumination light is further provided.

[0159] (4) A light path between the light source and the objective has areflection member provided so as to be capable of moving along the lightpath direction, and the restricting part restricts a movement range ofthe reflection member along the light path direction to the range wheretotal internal reflection illumination and approximate total internalreflection illumination can be obtained.

[0160] (5) An optical element which is insertably and removably arrangedto the light path between the light source and the objective anddiffuses the illumination light is further provided.

[0161] Moreover, the respective foregoing embodiments include thefollowing inventions, for example.

[0162] (1) A total internal reflection illumination apparatus applied toa microscope which illuminates a sample through an objective having anumerical aperture enabling the total internal reflection illumination,wherein the illumination light incident from the outside of themicroscope is reflected in a direction of an objective by a first totalinternal reflection mirror arranged in the vicinity of an outermost partof an observation optical path, the return light totally reflected onthe surface of the specimen is reflected by a second total internalreflection mirror arranged symmetrical with the first total internalreflection mirror so as to sandwich the observation optical axis and itis ended by light trapping part.

[0163] (2) In The total internal reflection illumination apparatusaccording to (1), wherein the light trapping part is arranged outsidethe microscope through an optical fiber.

[0164] (3) In The total internal reflection illumination apparatusaccording to (1), wherein the total internal reflection illuminationapparatus of the microscope can be integrally moved away from theobservation optical path, and the illumination light is turned on/off incooperation with insertion to or removal from the observation opticalpath.

[0165] The present invention is not restricted to each of the foregoingembodiments, and it can be modified on the embodying stage in many wayswithout changing its gist. In addition, the respective foregoingembodiments can be appropriately combined and applied.

[0166] Additionally, the foregoing embodiments include various kinds ofinventions, and a variety of inventions can be extracted fromappropriate combinations of a plurality of disclosed structuralrequirements. For example, even if some of the structural requirementsare deleted from all the structural requirements disclosed in theembodiments, the problems described in the section “problems to besolved by the invention” can be solved, and the structures obtained bydeleting the structural requirements can be extracted as inventions whenthe advantages described in the section “advantages of the invention”can be obtained.

[0167] According to each of the embodiments of the present invention, itis possible to provide the total internal reflection illuminationapparatus of the microscope which can constantly assure a goodfluorescence observation image.

[0168] As a result, the observation light beam of the fluorescencegenerated from the specimen can be set so as not to cross theillumination light beam reflected by the first total internal reflectionmirror on the observation optical path or the return light beam enteringthe second total internal reflection mirror, and the illumination lightbeam with which the specimen is irradiated and the return light beamafter total internal reflection can be set so as not to cross eachother.

[0169] Further, according to the embodiments of the present invention,since the return light beam reflected by the second total internalreflection mirror is positively removed to the outside of themicroscope, the possibility that the scattered light of the return lightbeam may permeate the observation optical path can be further reduced.

[0170] Furthermore, according to the present invention, since theillumination light can be turned on/off in cooperation with insertionand removal of the support member, the illumination light can beautomatically stopped when the support member is removed.

[0171] As a result, according to the present invention, the incidenceangle of the illumination light to the sample through the objective canbe constantly restricted to the range where the total internalreflection illumination and the approximate total internal reflectionillumination can be obtained. Therefore, color degradation of the entiresample due to the strong light of the incident-light fluorescenceillumination can be prevented, and the stable fluorescence observationbased on the total internal reflection illumination and the approximatetotal internal reflection illumination can be obtained. Furthermore,since the incidence angle of the illumination light to the sample fromthe objective can be constantly adjusted in the range of the totalinternal reflection illumination and the approximate total internalreflection illumination, the operability can be improved.

[0172] As described above, according to the present invention, it ispossible to provide the microscope which can realize the stablefluorescence observation based on the total internal reflectionillumination and the approximate total internal reflection illuminationand improve the operability.

[0173] Additional advantages and modifications will readily occur tothose skilled in the art. Therefore, the invention in its broaderaspects is not limited to the specific details and representativeembodiments shown and described herein. Accordingly, variousmodifications may be made without departing from the spirit or scope ofthe general invention concept as defined by the appended claims andtheir equivalents.

What is claimed is:
 1. A total internal reflection illuminationapparatus applied to a microscope which illuminates a sample through anobjective having a numerical aperture enabling total internal reflectionillumination, comprising: a first total internal reflection mirror whichis arranged in the vicinity of an outermost peripheral part of anobservation optical path of the microscope to reflect an incidentillumination light in a direction of the objective; a second totalinternal reflection mirror which is arranged at a symmetrical positionwith the first total internal reflection mirror to sandwich anobservation optical axis and reflects return light reflected on asurface of the sample in a direction different from the illuminationoptical path; and a return light dimming part configured to dim thereturn light reflected by the second total internal reflection mirror.2. The total internal reflection illumination apparatus according toclaim 1, wherein the return light dimming part has an external setportion which is set outside the microscope.
 3. The total internalreflection illumination apparatus according to claim 2, wherein thereturn light dimming part is connected to the external set portionthrough an optical fiber.
 4. The total internal reflection illuminationapparatus according to claim 1, wherein at least a part of the totalinternal reflection illumination apparatus is set to a support memberwhich insertable or removable to the observation optical path of themicroscope.
 5. The total internal reflection illumination apparatusaccording to claim 1, wherein the first total internal reflection mirrorand the second total internal reflection mirror are arranged atpositions where they are symmetrical with each other to sandwich anoptical axis of the objective, and the first total internal reflectionmirror and the second total internal reflection mirror move in such amanner a distance between the optical axis of the objective and thefirst total internal reflection mirror and a distance between theoptical axis of the objective and the second total internal reflectionmirror become equal to each other.
 6. The total internal reflectionillumination apparatus according to claim 4, wherein one of themicroscope and the support member has a switching part configured toturn on or turn off the illumination light in cooperation with insertionor removal of the support member.
 7. The microscope according to claim6, further comprising: a switching part configured to switch totalinternal reflection illumination and approximate total internalreflection illumination by changing an incidence angle of theillumination light with which the sample is irradiated; and arestricting part configured to restrict the incidence angle of theillumination light to the sample to a range where total internalreflection illumination and approximate total internal reflectionillumination can be obtained.
 8. A microscope comprising: a light sourcewhich emits predetermined light; a total internal reflectionillumination apparatus according to claim 1, which irradiates a samplewith the light from the light source through an objective; and an imagepickup device which images the light from the sample as an image.
 9. Themicroscope according to claim 8, wherein the light source has an opticalfiber having an outgoing end provided to be movable in a directionvertical to the optical axis, and the restricting part restricts amovement range of the outgoing end of the optical fiber in the directionvertical to the optical axis to the range where total internalreflection illumination and approximate total internal reflectionillumination can be obtained.
 10. The microscope according to claim 9,further comprising an optical element which is insertably and removablyarranged to the light path between the light source and the objectiveand diffuses the illumination light.
 11. The microscope according toclaim 8, wherein a light path between the light source and the objectivehas a reflection member arranged movably along a direction of the lightpath, and the restricting part restricts a movement range of thereflection member along the direction of the light path to the rangewhere total internal reflection illumination and approximate totalinternal reflection illumination can be obtained.
 12. The microscopeaccording to claim 11, further comprising an optical element which isinsertably and removably arranged to the light path between the lightsource and the objective and diffuses the illumination light.
 13. Amicroscope comprising: a changing part configured to change an incidenceangle of illumination light which is emitted onto a sample from a lightsource through an objective and enables switching between total internalreflection illumination and approximate total internal reflectionillumination; and a restricting part configured to restrict theincidence angle of the illumination light to the sample through theobjective to a range where total internal reflection illumination andapproximate total internal reflection illumination can be obtained. 14.The microscope according to claim 13, further comprising a dimming partconfigured to dim light reflected by the sample.
 15. The microscopeaccording to claim 13, wherein the light source has an optical fiberhaving an outgoing end provided to be movable in a direction vertical toan optical axis, and the restricting part restricts a movement range ofthe outgoing end of the optical fiber in the direction vertical to theoptical axis to the range where total internal reflection illuminationand approximate total internal reflection illumination can be obtained.16. The microscope according to claim 15, further comprising an opticalelement which is insertably and removably arranged the light pathbetween the light source and the objective and diffuses the illuminationlight.
 17. The microscope according to claim 13, wherein a light pathbetween the light source and the objective has a reflection memberprovided so as to be capable of moving along the light path direction,and the restricting part restricts a movement range of the reflectionmember along the light path direction to the range where total internalreflection illumination and approximate total internal reflectionillumination can be obtained.
 18. The microscope according to claim 17,further comprising an optical element which is insertably and removablyarranged to the light path between the light source and the objectiveand diffuses the illumination light.